Microplastic-induced intestinal injury alleviating postbiotic fermentation product of composite probiotics and application thereof

Abstract

The application provides a compound probiotic postbiotic fermentation product for relieving intestinal damage caused by microplastics, and an application thereof. The compound probiotic postbiotic fermentation product is prepared from fermentation of Lactobacillus plantarum HNU082, Lactobacillus fermentum HNU312 and Lactobacillus paracasei HNU502. The postbiotic prepared by the application can relieve inflammation induced by polystyrene, relieve reduction of Occludin and Claudin-1 expression induced by polystyrene, and relieve reduction of the number of colon goblet cells induced by polystyrene. Therefore, the postbiotic prepared by the application can relieve damage to the intestinal barrier of an organism caused by microplastics.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and relates to a compound probiotic postbiotic fermentation product for relieving intestinal damage caused by microplastics and application thereof. BACKGROUND

[0002] Microplastics are defined as plastic fragments or particles with a diameter of less than 5 mm. This concept was first proposed in Science in 2004, and the particle size of microplastics was clearly defined in 2008. Microplastics are divided into primary microplastics and secondary microplastics. Primary microplastics are larger plastic fragments or particles. Secondary microplastics are smaller plastic fragments or particles formed by a series of degradation and physical and chemical changes of primary microplastics in the environment. The United Nations Environment Programme (UNEP) estimates that the global production of primary plastics will reach 34 billion tons by 2050.

[0003] In today's life, plastic products are everywhere, such as plastic cups, bowls, plastic bags, food packaging bags, exfoliating scrubs in cosmetics, and cleaning mud masks. Human diet is also contaminated by microplastics. Fibers with a particle size of 13.45-6742.48 microns and fragments with a size of 2.48-247.54 microns were detected in Ecuadorian honey, beer, milk, and snacks. In addition, 16 types of microplastics were detected in placenta, meconium, baby feces, breast milk, and infant formula milk powder. There is also microplastic pollution in the environment, such as atmosphere, soil, seawater, freshwater, and land.

[0004] Researchers have found that exposure to microplastics can disrupt the stability of the intestinal microenvironment, especially the intestinal barrier, causing intestinal oxidative damage and inflammation, and damage to the intestinal epithelium, reduction of the mucus layer, and microbial disorder. SUMMARY

[0005] The purpose of the present application is to provide a compound probiotic postbiotic fermentation product for relieving intestinal damage caused by microplastics and application thereof, which is prepared by fermenting three strains of lactobacilli with coconut water to prepare postbiotics to relieve damage to the intestinal barrier caused by microplastics.

[0006] The technical scheme adopted by the present application to achieve the above-mentioned purpose is as follows:

[0007] The present application provides a compound probiotic postbiotic fermentation product for relieving intestinal damage caused by microplastics, which is prepared by fermenting Lactobacillus plantarum HNU082, Lactobacillus fermentum HNU312, and Lactobacillus paracasei HNU502 with coconut water.

[0008] The present application also provides a preparation method of a compound probiotic postbiotic fermentation product for relieving intestinal damage caused by microplastics, comprising:

[0009] Coconut water is filtered with gauze and sterilized at 115 DEG C for 15-20 min.

[0010] After the three generations of Lactobacillus plantarum HNU082, Lactobacillus fermentum HNU312 and Lactobacillus paracasei HNU502 are inoculated into sterilized coconut water and fermented, inactivation is performed to obtain a compound probiotic postbiotic fermentation product.

[0011] Preferably, the Lactobacillus plantarum HNU082, Lactobacillus fermentum HNU312 and Lactobacillus paracasei HNU502 are inoculated into MRS liquid medium respectively, and then placed in a 37 DEG C constant temperature incubator for culture for 24 h; 200 muL of the bacterial liquid is subcultured in MRS liquid medium, and then placed in a 37 DEG C constant temperature incubator for culture for 24 h; 200 muL of the bacterial liquid is subcultured in MRS liquid medium again, which is the third generation of bacteria; the bacterial liquid of the third generation of bacteria is centrifuged at 4000 r for 5 min, and the supernatant is discarded; physiological saline is added and mixed, and then sterilized coconut water is added for fermentation culture at 37 DEG C for 16 h.

[0012] Preferably, each strain is inoculated into sterilized coconut water at a inoculation amount of 3%, so that the inoculation amount of the mixed bacteria is 9%.

[0013] Preferably, the fermentation liquid is inactivated by a high-pressure steam sterilization pot at 95-105 DEG C for 15-20 min.

[0014] The application further provides application of the compound probiotic postbiotic fermentation product in preparation of a product for relieving damage of microplastics to intestinal tracts of organisms.

[0015] Preferably, the compound probiotic postbiotic fermentation product relieves damage of microplastics to intestinal tracts of organisms by relieving inflammation induced by polystyrene.

[0016] More preferably, the compound probiotic postbiotic fermentation product relieves inflammation induced by polystyrene by reducing production of proinflammatory factors IFN-gamma and IL-6 and relieving inflammatory factors.

[0017] Preferably, the compound probiotic postbiotic fermentation product relieves damage of microplastics to intestinal tracts of organisms by relieving reduction of occludin and claudin-1 expression induced by polystyrene.

[0018] Preferably, the compound probiotic postbiotic fermentation product relieves damage of microplastics to intestinal tracts of organisms by relieving reduction of the number of colon goblet cells induced by polystyrene.

[0019] The application has the following beneficial effects:

[0020] The present application prepares postbiotics by fermenting three strains of lactobacillus with coconut water. Coconut water mainly contains glucose and fructose, and is rich in nutrients, containing more than 17 free amino acids such as arginine, alanine, cystine and serine, vitamin C and B vitamins, and minerals, among which the content of minerals Fe 2+ and K + is the highest, which is a natural culture medium for cultivating microorganisms. At the same time, coconut water also has the effects of beautifying and nourishing the skin, diuresis and detumescence, sterilization and anti-inflammation, and maintaining the stability of intestinal flora in the human body. Compared with probiotics, postbiotics is not a living microorganism, has a longer shelf life, is safer, is also suitable for some special groups of people, has no risk of transmitting drug-resistant genes, has a wider target for action, and is easier to be absorbed.

[0021] The postbiotics prepared by the present application can relieve the inflammation induced by polystyrene, relieve the decrease in the expression of occludin and claudin-1 induced by polystyrene, and relieve the decrease in the number of colon goblet cells induced by polystyrene, so the postbiotics prepared by the present application can relieve the damage of microplastics to the intestinal barrier of the body. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 (A) IL-6 (B) IFN-gamma (C) AST (D) ALT.

[0023] Figure 2 (A) Representative immunofluorescence pictures of TJs proteins in the colon of each group (red represents tight junction proteins); (B) Claudin-1 immunofluorescence protein positive area ratio; (C) Occludin immunofluorescence protein positive area ratio.

[0024] Figure 3 (A) AB-PAS staining of the colon of mice; (B) Number of AB-PAS positive cells in the colon of mice. DETAILED DESCRIPTION

[0025] In order to more clearly illustrate the present application, the present application will be further described in detail below in conjunction with the embodiments and by referring to the accompanying drawings. Those skilled in the art should understand that the specific description below is illustrative rather than limiting, and should not limit the protection scope of the present application.

[0026] EMBODIMENTS

[0027] I. Preparation of postbiotics

[0028] Fresh coconut water was filtered with 100 mesh gauze, and then was filled into conical flask and sterilized at 115℃ for 15-20 min for standby. The three strains of Lactobacillus used in this experiment were Lactobacillus plantarum HNU082 (preserved number GDMCC NO: 61552), Lactobacillus fermentum HNU312 (preserved number GDMCC NO: 62803), and Lactobacillus paracasei HNU502 (the strain was isolated and screened from infant feces in the laboratory, and was preserved in Guangdong Microbial Culture Collection Center on September 14, 2023, with the preserved number GDMCC No: 63813). The frozen Lactobacillus was inoculated into a glass test tube containing 5 mL of MRS liquid medium, and then was placed in a 37℃ constant temperature incubator for 24 h. 200 μL of bacterial liquid was subcultured in MRS liquid medium, and was placed in a 37℃ constant temperature incubator for 24 h. Then, 200 μL of bacterial liquid was subcultured in MRS liquid medium again, which was the third generation of bacteria. Each strain was inoculated into sterilized coconut water at a concentration of 3%, and the mixed bacterial inoculation amount was 9%. After centrifugation at 4000 r for 5 min, the supernatant was discarded, 1 mL of normal saline was added and mixed, and then the mixture was added into sterilized coconut water and cultured at 37℃ for 16 h. Then, the fermentation liquid was inactivated by high-pressure steam sterilization pot at 105℃ for 20 min.

[0029] II. Animal experiment

[0030] Statistical analysis was performed using R (version 3.6.1) software and SPSS 26. Data were expressed as mean ± standard deviation (SD). Differences were considered significant at a p-value threshold of 0.05 (*p<0.05, **p<0.01, ***p<0.001). GraphPad Prism 8 was used for plotting.

[0031] 1. Experimental materials and animal experiment design

[0032] Polystyrene with a particle size of 5 μm (PS-MPS) was purchased from Tianjin Baisi Le Chromatography Technology Development Center, and the mother liquor concentration was 25 mg / mL. Before the experiment, the particle mother liquor was ultrasonically treated for 10 minutes. The PS-MPS suspension used during the experiment was prepared by diluting the purchased PS-MPS stock solution with deionized water to the desired concentration, and then shaking and ultrasonic treatment were performed to ensure that the PS-MPS was uniformly dispersed in the deionized water.

[0033] 6-week-old C57BL / 6J male mice 24, after 7 days of normal diet adaptation in the laboratory environment, were randomly divided into 4 groups, control group, model group, probiotics group, coconut group, once a day, the first time, the control group was gavaged with 300 μL of deionized water; the model group was gavaged with 300 μL of polystyrene microplastic suspension; the probiotic group was gavaged with 100 μL of polystyrene microplastic suspension and 200 μL of probiotic; the coconut group was gavaged with 100 μL of polystyrene microplastic suspension and 200 μL of coconut water. Except for the control group, the remaining three groups of microplastic exposure dose were 10 mg / kg. One week of gavage for 6 days, after continuous gavage for 8 weeks, the mice were fasted for 12 h, the eyeball was taken, and the mouse was dissected after the neck was broken, the kidney, liver, spleen, epididymal fat, colon, colon contents, cecum contents, ileum, ileum contents, the samples were stored at -80℃ until further use. After the mouse blood was left to stand for 2-3 h, it was centrifuged at 3200 r / 10 min, the supernatant was collected and placed in a -80℃ refrigerator.

[0034] 2. Serum index determination

[0035] The levels of IL-6 and IFN-γ in serum were determined by ELISA kit. The levels of AST and ALT in serum were determined by Nanjing Jiancheng kit.

[0036] Long-term exposure to microplastics can cause inflammation in the body. From Figure 1 (A) and (B) can be seen, compared with the control group, the model group pro-inflammatory factors IFN-γ, IL-6 increased significantly, and probiotics can alleviate the production of inflammatory factors.

[0037] 3. Immunohistochemistry and immunofluorescence staining

[0038] Tight junction (TJ) is an important structure that constitutes the function of epithelial barrier, which plays a decisive role in intestinal mucosa permeability, and is an important part of intestinal mechanical barrier. Long-term exposure to microplastics can induce the destruction of intestinal physical intestinal barrier, mainly manifested as the decrease of TJ proteins such as Occludin and Claudins in intestinal epithelial cells. In order to explore the effect of PS-MPS on intestinal barrier, immunohistochemical method was used to observe the distribution and structural changes of TJ related proteins, Occludin and Claudin-1 proteins.

[0039] Paraffin-embedded tissue sections (4 μm) were deparaffinized with xylene and rehydrated in a graded alcohol series. After permeabilization with 0.5% Triton-X 100 for 15 min and antigen retrieval in citrate buffer, the sections were blocked for endogenous peroxidase activity with 3% H2O2 and blocked with goat serum. The sections were then incubated with antibodies overnight at 4°C. Next, the sections were washed 5 times for 3 min each with PBS and treated with 3,3'-diaminobenzidine tetrahydrochloride for 15 min. For immunofluorescence, the tissues were stained with DAPI. The tissues were observed under a light microscope.

[0040] As shown in Figure 2 Fig. 2B, compared with the control group, the expression of Occludin and Claudin-1 in the model group was significantly reduced, and the postbiotic group could alleviate the reduction of the expression of Occludin and Claudin-1 induced by polystyrene.

[0041] 4. Histopathological analysis

[0042] Intestinal epithelial integrity is considered the first line of defense of the gastrointestinal tract. The effect of polystyrene exposure on intestinal mucin secretion was observed by AB-PAS staining of the colon. The colon was cut into small pieces and immediately fixed in a 10% formaldehyde solution. Subsequently, the fixed intestinal tissue was dehydrated in a series of ascending ethanol, cleared in xylene, and embedded in paraffin at 56°C. Then, three midgut samples of each group were cut into 5-μm-thick sections. The sections were stained with Alcian blue-periodic acid-Schiff (AB-PAS) solution, and the staining results are shown in Figure 3 (A).

[0043] As shown in Figure 3 (B), compared with the control group, the number of goblet cells was significantly reduced, and the postbiotic group could alleviate the reduction of the number of colon goblet cells induced by polystyrene.

[0044] Obviously, the above embodiments of the present application are only examples for more clearly illustrating the present application, and are not intended to limit the embodiments of the present application. On the basis of the above description, other different forms of changes or variations can be made by those skilled in the art, and it is impossible to enumerate all the embodiments here. Any obvious changes or variations derived from the technical solutions of the present application are still within the protection scope of the present application.

Claims

1. A compound probiotic postbiotic ferment for relieving intestinal damage caused by microplastics, which is prepared by fermenting Lactobacillus plantarum HNU082, Lactobacillus fermentum HNU312 and Lactobacillus paracasei HNU502 in coconut water.

2. A method for preparing a compound probiotic postbiotic ferment for relieving intestinal damage caused by microplastics, comprising the following steps: sterilizing coconut water at 115°C for 15-20 min after filtering it with gauze; and fermenting and culturing Lactobacillus plantarum HNU082, Lactobacillus fermentum HNU312 and Lactobacillus paracasei HNU502 in the sterilized coconut water, inactivating the three generations of bacteria to obtain the compound probiotic postbiotic ferment. Inoculating Lactobacillus plantarum HNU082, Lactobacillus fermentum HNU312 and Lactobacillus paracasei HNU502 in MRS liquid medium respectively, and then culturing them in a 37°C constant-temperature incubator for 24 h; subculturing 200 µL of the bacterial liquid in MRS liquid medium, and then culturing it in a 37°C constant-temperature incubator for 24 h; subculturing 200 µL of the bacterial liquid in MRS liquid medium again, which is the third generation of bacteria; centrifuging the bacterial liquid of the third generation of bacteria at 4000 r for 5 min, discarding the supernatant, mixing with physiological saline, and then adding the sterilized coconut water to ferment and culture it at 37°C for 16 h. Inoculating each strain of bacteria in the sterilized coconut water at a 3% inoculation amount, so that the inoculation amount of the mixed bacteria is 9%.

3. The production method according to claim 2, characterized by, Inactivating the fermentation liquid by using a high-pressure steam sterilization pot at 95-105°C for 15-20 min.

4. The production method according to claim 2 or 3, characterized by, 6. Use of the compound probiotic postbiotic ferment of claim 1 in the preparation of a medicament for relieving intestinal damage caused by microplastics.

5. The preparation method according to claim 2, characterized in that, The compound probiotic postbiotic ferment relieves the damage to the intestinal barrier of the body caused by microplastics by relieving inflammation induced by polystyrene. The compound probiotic postbiotic ferment relieves inflammation induced by polystyrene by reducing the production of pro-inflammatory factors IFN-γ and IL-6 and anti-inflammatory factors in the serum.

7. Use according to claim 6, characterized in that, The compound probiotic postbiotic ferment relieves the damage to the intestinal barrier of the body caused by microplastics by relieving the decrease in the expression of Occludin and Claudin-1 induced by polystyrene.

8. Use according to claim 7, characterized in that, The compound probiotic postbiotic ferment relieves the damage to the intestinal barrier of the body caused by microplastics by relieving the decrease in the number of colon goblet cells induced by polystyrene.

9. Use according to claim 6, characterized in that, ​ 10. Use according to claim 6, characterized in that, ​

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